1. Field of the Invention
The present invention relates to a method for producing human insulin-like growth factor I (hereinafter abbreviated as hIGF-I). Specifically, it relates to a method for producing hIGF-I whereby hIGF-I can be obtained with high purity and yield by letting a culture liquid of a hIGF-I producing bacteria stand under specific conditions.
2. Description of the Related Art
hIGF-I is a known polypeptide compound existing in nature (E. Rinderknecht et al., “The amino acid sequence of human insulin-like growth factor I and its structural homology with proinsulin”, J. Biol. Chem., 1978, 253(8): p. 2769-76; M. Iwai et al., “Direct identification of disulfide bond linkages in human insulin-like growth factor I (IGF-I) by chemical synthesis”, J. Biochem. (Tokyo), 1989, 106(6): p. 949-51) and having cell proliferation activity (R. E. Humbel, “Insulin-like growth factors I and II”, Eur. J. Biochem., 1990, 190(3): p. 445-62).
hIGF-I can itself be used as a drug, and drug applications are being developed (see for example M. O. Savage et al., “Therapeutic applications of the insulin-like growth factors”, Growth Horm. IGF Res., 2004, 14(4): p. 301-8; L. Gasparini et al., “Potential roles of insulin and IGF-I in Alzheimer's disease”, Trends Neurosci., 2003, 26(8): p. 404-6; J. Ren et al., “Insulin-like growth factor I as a cardiac hormone: physiological and pathophysiological implications in heart disease”, J. Mol. Cell. Cardiol., 1999, 31(11): p. 2049-61). Because hIGF-I has cell proliferation activity, it can be also used in the production of antibody drugs and other biotechnology-based drugs as a medium additive for cells producing the proteins that are the active components of these drugs. Since production of biotechnology-based drugs is rising rapidly, demand for large quantities of hIGF-I is anticipated.
Under these circumstances, various methods have been reported for producing and refining hIGF-I by culturing transformed microorganisms by recombinant DNA methods (hereinafter referred to as recombinant microorganisms). For example, recombinant E. coli is used in U.S. Pat. No. 6,331,414, and recombinant yeast is used in U.S. Pat. No. 5,324,639.
In these methods, the recombinant E. coli or recombinant yeast is cultured to produce hIGF-I and the resulting hIGF-I is purified. Pigments, proteins and the like that are intrinsically produced by the E. coli or yeast also occur along with the target hIGF-I in the hIGF-I culture process and/or purification process, as does isomers having a different primary structure from hIGF-I.
One of the isomers, which is called misfolded hIGF-I because the combination of three pairs of disulfide bonds formed within the molecule differs from that of natural hIGF-I, is known to differ from natural hIGF-I not only in its physical properties but also in its biological properties (A. Sato et al., “Three-dimensional solution structure of a disulfide bond isomer of the human insulin-like growth factor-I”, J. Pept. Res., 2000, 56(4), p. 218-30). Consequently, this isomer must be isolated and removed from the natural hIGF-I. Methods that have been reported for doing this include a method of isomerizing the isomer into natural hIGF-I and a method of isolating and removing the isomer (see for example U.S. Pat. Nos. 7,071,313; 5,231,178).
For industrial purposes, there need to be methods of producing hIGF-I at low cost and in large volume.
The applicant of the application previously discovered that when a recombinant coryneform bacterium is used as the host for producing human epithelial cell growth factor, the amount of intrinsic proteins produced in the culture liquid of the recombinant coryneform bacterium is less than the amount of intrinsic proteins produced in the culture liquid of other recombinant microorganisms (JP 2002-291476 A).